Compound, especially marker-dye on the basis of polymethines

ABSTRACT

The invention relates to fluorescent dyes (fluorophores) based on polymethines for use in optical measurement and detection procedures, in particular those employing fluorescence, for example in medicine, in pharmacology and in the biological, materials and environmental sciences. The objective was to create fluorophores based on polymethines that have a large Stokes shift, high photostability, long storage life and a high fluorescent quantum yield, and that can be excited in the simplest possible manner by white-light sources or laser radiation in the UV, visible or NIR spectral region. According to the invention dyes on the basis of polymethines having the general formulas I, II or III  
                 
 
     are employed.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation-in-part of application Ser.No. 10/310,206, filed Dec. 5, 2002 [Attorney Docket No. 39159-P003US],by inventors Czerney, et al., currently pending.

[0002] This application also claims priority under the Paris Conventionto DE application Serial No. 103 56 130.7, filed with the German PatentOffice on Nov. 28, 2003.

BACKGROUND OF INVENTION

[0003] Field of Invention

[0004] The invention relates to fluorescent dyes (fluorophores) based onpolymethines for use in optical measurement and detection procedures, inparticular those employing fluorescence. Typical applications exploitthe reaction of dye-labelled antigens, antibodies or DNA segments withthe respective complementary species. With such methods it is possibleto measure, e.g., enzyme kinetics, receptor-ligand interactions and thekinetics of nucleic-acid hybridization. Furthermore, the claimedfluorophores are of interest for the pharmacological characterization ofreceptors or agents.

[0005] Possible uses associated therewith exist, for example, inmedicine and pharmacology, in the biological and materials sciences, inenvironmental monitoring and the detection of organic and inorganicmicrosamples present in natural surroundings and in technology.

[0006] Whereas cyanines with a Stokes shift of 20-40 nm have long beenknown as fluorescent markers (Cy3, Cy5, U.S. Pat. No. 5,627,027), as yetthere are only a few fluorophores with a large Stokes shift. Typicalexamples of these are the markers derived from the laser dye DCM, withabsorption maxima at 481 nm and emission maxima at 644 nm, which areclaimed in the patent U.S. Pat. No. 4,886,744.

[0007] Different stilbenes with large Stoke's shift have been previouslydescribed by F. Lehmann et al. in “Dyes and Pigments”, Elsevier AppliedScience Publishers, vol. 29, no. 1, 1995, pages 85-94 as well as by G.J. Ashwell et al. in the “Journal of Materials Chemistry”, Cambridge,vol. 11, no. 5, 2001, pages 1345-50. Hydroxy substituted-stilbenes witha lactone bridging have been published by R. M. Abd El-Aal et al. in the“Journal of the Chinese Chemical Society”, Taipei, vol. 47, no. 2, 2000,pages 389-95. Further to be taken into consideration are the differentstilbenes published by Czerney et al. in “Sensors and Actuators B”,Elsevier, Lausanne, vol. 39, no. 1-3, 1997, pages 395-400 and in U.S.Pat. No. 6,096,794.

[0008] Common to all compounds is that they do not have any reactivegroup which permits covalent binding, for example, biomolecules. Afurther disadvantage is that the solubility in water or aqueous mediafor applications in bioanalysis is not sufficient.

[0009] Whereas the compounds disclosed in the Japanese unexamined patentspecification JP 07-234 504 contain an —SO₃—group, they have acompletely different structure than the compounds I to III according tothe invention. They are distinguished from the latter in that they arenot suited for applications in bioanalysis and diagnosis because theylack a function which enables a covalent binding on biomolecules.

SUMMARY OF INVENTION

[0010] The invention is directed towards the objective of creatingfluorescent markers on the basis of polymethines that have an adjustablelarge Stokes shift which can be adjusted as desired to the requirementsof the optical detection system, with high photostability, long storagelife and a high fluorescence quantum yield. The compunds of theinvention have the advantage that the Stokes shift can be influenced bythe kind of the substituents and the value of m. They are excellentlysuitable for multicolor applications. Moreover, such fluorescent markersdo not only have a large Stokes shift, but are also characterized by ahigh fluorescence quantum yield in the red spectral range. Therefore,they are characterized by having the advantages of fluorescent markerswith a high fluorescence quantum yield in the red spectral range (e.g.Cy-5, DY-630) and those having a large Stokes shift. In addition, thefluorescence quantum yield of the present compounds is sensitive towardsproteins so that they are excellently suitable for dyeing proteins(proteinstain) in a non-covalent manner. To achieve an optimalsignal-to-noise ratio, the emission bands should be in a range >520 nm,and the excitation should be achievable in the simplest possible way, bywhite light or laser radiation in the UV or visible spectral region. Thefluorophores must have a high fluorescence output, independent of the pHvalue and other environmental influences. A prerequisite for covalentbinding is the presence of a reactive function such that the reactionwith the biomolecule to be labelled occurs under physiologicalconditions, or under the reaction conditions customary in thesolid-phase synthesis of bio-oligomers.

[0011] The present invention describes polymethine-based compounds,particularly marker dyes with the general formulas I or II or III:

[0012] where

[0013] R¹-R¹² are the same or different and can be any of the following:hydrogen, one or more chlorine and/or bromine atoms, alkyl, aryl,heteroaryl, cycloalkyl, heterocycloalkyl, alkyloxy, alkylmercapto,aryloxy, arylmercapto, heteroaryloxy, heteroarylmercapto or cyanoresidues, one or more alkyl-substituted or cyclic amino functions,preferably those which have at most 12 carbon atoms, one or more hydroxyfunctions,

[0014] R¹ and R² and/or R³ and R⁴ and/or R⁷ and R⁸ and/or R⁹ and R¹⁰ canform additional aliphatic or aromatic rings, and/or two adjacentresidues (e.g. R⁵ and R⁶, and in examples I or II R¹¹ and R¹²), togethercan form one or more aliphatic or aromatic rings, preferably at most tworings,

[0015] one or more of the substituents R¹-R¹² can be solubilizing orionizable or ionized substituents such as SO₃ ⁻, PO₃ ⁻, CO₂ ⁻, O⁻, NR₃⁺, cyclodextrin or sugar, which determine the hydrophilic properties ofthe dyes, and it is also possible for these substituents to be bound tothe actual chromophore via an aliphatic or heteroaliphatic, possiblycyclic spacer group,

[0016] at least one of the substituents R¹-R¹² stands for a reactivegroup of the type isocyanate, isothiocyanate, hydrazine,amine^(, mono- and dichloro- or mono- and dibromotriazine, aziridine, sulfonyl halide, N-hydroxysuccinimide ester, imido ester, glyoxal or aldehyde, or maleimide or iodacetamide and phosphoramidite, and the substituent in each case being unchanged or bound to the actual basic chromophore via an aliphatic or heteroaliphatlc, possibly cyclic spacer group,)

[0017] X-Y together can stand for an element of the group O, S, Se, Teor the structural element (CR₂)_(n), NR or SO₂, wherein R can take onthe same one or different ones of the functions of R¹-R¹², and n canstand for 1-4,

[0018] X-Y can stand for the structural elements —CR₂—O—, —O—CR₂—,—CO—O—, —O—CO—, —CO—NR— or —NR—CO—, wherein R can take on the same oneor different ones of the functions of R¹-R¹²,

[0019] Z represents the group (CR₂)_(p), wherein R represents equal ordifferent groups of R₁ to R₁₂, —(CH₂)_(r)—COOH or —(CH₂)_(r)—SO₃H ortheir dissociable salts, p is an integer of 1 to 4 and r is an integerof 1 to 7, preferably 1 to 5, or a combination of any of these groups,and

[0020] m is zero or an integer from 1 to 3.

[0021] In preferred compounds of the invention m is 1 or Z is C(CH₃)₂ orm is 1 and Z is C(CH₃)₂. Moreover, compounds are preferred in which X-Ytogether represent —CO—O— or —O—. In other preferred compounds R⁵ is H,OH or alkoxy, preferably with at most 4 carbon atoms, and/or R¹ iscarboxyalkyl, the carboxy group being preferably in o-position.Moreover, compounds are preferred wherein one of R⁸ to R¹ is or containsa SO₃ ⁻ group or R² is an amino residue, bound to the nucleus by anitrogen atom which may contain substituents of at most 8 carbon atomsincluding a carboxyalkyl or a sulfonic acid alkyl. In specific compoundsof this type, R² may also form a ring with R¹ and/or R³. Moreover,compounds are preferred in which a caboxyalkyl group, being present asR¹¹ or contained in R², is an co-carboxyalkyl having 6 carbon atoms.Other preferred compounds are those in which the aliphatic orheteroaliphatic spacer group consists of a structural element—[(CH₂)_(a)—W— (CH₂)_(b)]_(c)—, wherein W can be the same or differentones of the functions CR₂, O, S, SO₂, SO₂NH, NR, COO, or CONR, R cantake on the functions of R¹-R¹², and a and b represent the same ordifferent values in the range 0-18, while c represents the values from 1to 18.

[0022] The substituted polymethine derivatives with the general formulasI-III can be used as dyes for the optical marking of proteins, nucleicacids, oligomers, DNA, RNA, biological cells, lipids, mono-, oligo- andpolysaccharides, ligands, receptors, polymers, pharmaceuticals orpolymer particles. In order to serve as dyes in systems for thequalitative or quantitative determination of proteins, nucleic acids,oligomers, DNA, RNA, biological cells, lipids, polymers, pharmaceuticalsor polymer particles, the substituted polymethine derivatives with thegeneral formulas I-III are coupled by way of the functional groups to anHO—, H₂N— or HS-function of the substances to be determined, thecoupling reaction of the substituted polymethine derivatives of thegeneral formula I-III suitably being carried out in an organic oraqueous medium, e.g. a dispersion or suspension, but preferably asolution, to yield conjugates comprising substituted polymethinederivatives with the general formulas I-III and biomolecules exhibitingfluorescent properties.

[0023] The substituted polymethine derivatives with the general formulasI-III and the systems derived therefrom can be employed with advantagein qualitative and quantitative optical measurement procedures, inparticular those based on fluorescence, including immunological tests,hybridization procedures, processes yielding DNA-sequences,chromatographic or electrophoretic methods and high-throughput screeningand for the analysis of receptor-ligand interactions on a microarray.

[0024] The polymethines with the general formulas I-III can be used asdyes for the optical marking of organic or inorganic identificationunits, e.g. amino acids, peptides, proteins, antigens, haptens, enzymesubstrates, enzyme cofactors, biotin, carotinoids, hormones,neurohormones, neurotransmitters, growth factors, lympholocines,lectins, toxins, carbohydrates, oligosaccharides, polysaccharides,dextrans, nucleic acids, oligonucleotides, DNA, RNA, biological cells,lipids, receptor-binding pharmaceuticals or organic or inorganicpolymeric carrier materials.

[0025] This marking of the identification units can be achieved by theproduction of ionic interactions between the markers having the generalformulas I-III and the materials to be marked.

[0026] Furthermore, it is also possible to produce a covalent bonding ofthe identification unit or the carrier material to the fluorophore. Thiscoupling reaction can be carried out in aqueous or predominantly aqueoussolution and preferably at ambient temperature. By this means afluorescent probe (conjugate) is produced that can be used for thequalitative or quantitative determination of various biomaterials orother organic and inorganic materials.

[0027] Both the compounds with the general formulas I-III and systemsderived therefrom can be employed in qualitative and quantitativeoptical measurement procedures, in particular those based onfluorescence, for the diagnosis of cell properties, in biosensors(point-of-care measurements), for research on the genome (DNAsequencing) and in miniaturization technologies. Typical applicationscan be found in cytometry and cell-sorting, fluorescence-correlationspectroscopy (FCS), in ultra-high-throughput screening (UHTS), inmulticolor fluorescence in situ hybridization (FISH), as proteinstainand in microarrays (DNA and protein chips).

[0028] Such a microarray is a raster-like arrangement of moleculesimmobilized on at least one surface, which can be used to studyreceptor-ligand interactions. The term “raster-like arrangement”signifies more than two molecules that are different from one another,are situated within a specified area, and in that area are immobilizedin different, prespecified regions with known positions.

[0029] A receptor is a molecule that has an affinity for a given ligand.Receptors can be naturally occurring or artificially produced molecules.Receptors can be used in pure form or while bound to other species.Receptors can be linked to a binding partner by covalent or non-covalentbonds, either directly or by way of particular coupling mediators.

[0030] Examples of receptors that can be detected by means of thisinvention include agonists and antagonists for cell-membrane receptors,toxins and other poisonous substances, viral epitopes, hormones such asopiates and steroids, hormone receptors, peptides, enzymes, enzymesubstrates, agents that function as cofactors, lectins, sugars,oligonucleotides, nucleic acids, oligosaccharides, cells, cellfragments, tissue fragments, proteins and antibodies, but are notlimited to the substances listed here.

[0031] A ligand is a molecule that is recognized by a particularreceptor. Examples of ligands that can be detected by this inventioninclude agonists and antagonists for cell-membrane receptors, toxins andother poisonous substances, viral epitopes, hormones such as opiates andsteroids, hormone receptors, peptides, enzymes, enzyme substrates,agents that function as cofactors, lectins, sugars, oligonucleotides,nucleic acids, oligosaccharides, proteins and antibodies, but are notlimited to the substances listed here.

[0032] By the preparation of asymmetrical polymethines, which compriseon one hand, as a terminal function, a readily derivatizable heterocycleof the type CH-acid compounds, and on the other hand a novel substituted6-ring heterocycle, in particular the following advantages are achieved:

[0033] With suitable substitution, the absorption maxima are in theregion of the emission wavelengths of the argon-ion laser (488 nm) or ofthe conventional laser diodes at 630-730 nm, and the emission maximaobserved are in the ranges between 510 and 710, particularly 550 and 660nm, or 660 and 820 nm, respectively.

[0034] Another advantage resides in the fact that the hydrophiliccharacter of the differently emitting fluorophores can be made nearlyidentical.

[0035] General directions for producing the dyes of Examples 1 to 15.

[0036] 0.2 mmol of the substituted coumarin-3-carbaldehyde, or 0.2 mmolof the benzofuran-2-carbaldehyde, and 0.2 mmol of the correspondingCH-active compound are heated with reflux in 5 ml acetic anhydride for10 hours. After cooling diethyl ether is added and the resultingprecipitate is isolated and purified by column chromatography.

EXAMPLE 1

[0037]1-(5-carboxy-pentyl)-2-[(E)-2-(7-diethylamino-2-oxo-2H-chromen3-yl)-vinyl]-pyridiniumbromide

[0038] 0.2 mmol 7-diethylamino-2-oxo-2H-chromen-3-carbaldehyd and 0.2mmol 1-(5-carboxy-pentyl)-2-methyl-pyridinium bromide are convertedaccording to the general directions above. Column chromatography: SiO₂,eluent: ethanol/toluene.

[0039] 35% yield, MS (ESI+):435 (M⁺), UV-Vis (in ethanol): λ_(max) 480nm, λ_(em) 600 nm

EXAMPLE 2

[0040]1-(5-carboxy-pentyl)-2-[(E)-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-vinyl]-5-sulfonato-pyridiniumbetaine

[0041] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-2-methyl-5-sulfonato-pyridinium betaine areconverted according to the general directions above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water. 30% yield, MS(ESI+): 537 (M+Na⁺), UV-Vis (in water): λ_(max) 505 nm, λ_(em) 640 nm

EXAMPLE 3

[0042]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-vinyl]-pyridiniumbromide

[0043] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-4-methyl-pyridinium bromide are convertedaccording to the general directions above. Column chromatography: SiO₂,eluent: ethanol/toluene. 42% yield, MS (ESI+): 435 (M⁺), UV-Vis (inethanol): λ_(max) 500 nm, λ_(em) 630 nm

EXAMPLE 4

[0044]1-(5-carboxy-pentyl)-4-((E)-2-{7-[ethyl-(3-sulfonatopropyl)-amino]-2-oxo-2H-chromen-3-yl}-vinyl)-pyridiniumbetaine

[0045] 0.2 mmol3-[ethyl-(3-formyl-2-oxo-2H-chromen-7-yl)-amino]-propane-1-sulfonic acidsodium salt and 0.2 mmol 1-(5-carboxy-pentyl)-4-methyl-pyridiniumbromide are converted according to the general directions above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water.

[0046] 28% yield, MS (ESI—): 527 (M−H⁺), UV-Vis (in water): λ_(max) 480nm, λ_(em) 624 nm

EXAMPLE 5

[0047]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-vinyl]-3-sulfonato-pyridiniumbetaine

[0048] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-3-sulfonato-4-methyl-pyridinium betaine areconverted according to the general directions above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water. 30% yield, MS(ESI+): 537 (M−Na⁺), UV-Vis (in water): λ_(max) 524 nm, λ_(em) 660 nm

EXAMPLE 6

[0049]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-4-methoxy-2-oxo-2H-chromen-3-yl)-vinyl]-3-sulfonato-pyridinium betaine

[0050] 0.2 mmol7-diethylamino-4-(1-morpholino)-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-3-sulfonato-4-methyl-pyridinium betaine areconverted in methanol in the presence of triethylamine. Columnchromatography: SiO₂ (RP18), eluent: methanol/water.

[0051] 10% yield, MS (ESI+): 567 (M−Na⁺), UV-Vis (in water): λ_(max) 505nm, λ_(em) 645 nm

EXAMPLE 7

[0052]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-4-hydroxy-2-oxo-2H-chromen-3-yl)-vinyl]-3-sulfonato-pyridinium betaine

[0053] 0.2 mmol7-diethylamino-4-(1-morpholino)-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-3-sulfonato-4-methyl-pyridinium betaine areconverted in a methanol/water mixture in the presence of triethylamine.Column chromatography: SiO₂ (RP 18), eluent: methanol/water.

[0054] 15% yield, MS (ESI−): 529 (M−H⁺), UV-Vis (in water): λ_(max) 500nm, λ_(em) 590 nm

EXAMPLE 8

[0055]1-(5-carboxy-pentyl)-2-[(E)-2-(7-diethylamino-4-hydroxy-2-oxo-2H-chromen-3-yl)-vinyl]-3,3-dimethyl-5-sulfonato-3H-indolium betaine

[0056] 0.2 mmol 7-diethylamino-4-hydroxy-2-oxo-2H-chromene and 0.2 mmol1-(5-carboxy-pentyl)-3,3-dimethyl-2-((E)-2-phenylamino-vinyl)-5-sulfonato-3H-indoliumbetaine are converted according to the general directions givenhere-below as method 3. Column chromatography: SiO₂ (RP 18), eluent:methanol/water.

[0057] 35% yield, MS (ESI−): 595 (M−H⁺), UV-Vis (in water): λ_(max) 505nm, λ_(em) 600 nm

EXAMPLE 9

[0058]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-vinyl]-chinoliniumbromide

[0059] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-4-methyl-chinolinium bromide are convertedaccording to the general directions given above.

[0060] Column chromatography: SiO₂, eluent: ethanol/toluene. 30% yield,MS (ESI+): 485 (M⁺), UV-Vis (in methanol): λ_(max) 540 nm, λ_(em) 695 nm

EXAMPLE 10

[0061]1-(5-carboxy-pentyl)-4-[(E)-2-(7-diethylamino-2-oxo-2H-chromen-3-yl)-vinyl]-6-sulfo-chinoliniumbetaine

[0062] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-4-methyl-6-sulfonato-chinolinium betaine areconverted according to the general directions given above. Columnchromatography: SiO₂ (RP 18), eluent: methanol/water.

[0063] 20% yield, MS (ESI+): 465 (M+H⁺) and 587 (M+Na⁺), UV-Vis (inmethanol): λ_(max) 555 nm, λ_(em) 715 nm

EXAMPLE 11

[0064]1-(5-carboxy-pentyl)-2-[(E)-2-(7-diethylamino-2-oxo-2H-chromen1-3-yl)-vinyl]-6-methyl-chinoliniumbromide

[0065] 0.2 mmol 7-diethylamino-2-oxo-2H-chromene-3-carbaldehyde and 0.2mmol 1-(5-carboxy-pentyl)-4,6-dimethyl-chinolinium bromide are convertedaccording to the general directions given above. Column chromatography:SiO₂ (RP18), eluent: methanol/water.

[0066] 10% yield, MS (ESI+): 499 (M⁺), UV-Vis (in methanol): λ_(max) 520nm, λ_(em) 655 nm

EXAMPLE 12

[0067] 1-(5-carboxy-pentyl)-2-[(E)-2-(6-diethylamino-benzofuran-2-yl)vinyl]-5-sulfonato-pyridinium betaine

[0068] 0.2 mmol 6-diethylamino-benzofuran-2-carbaldehyde and 0.2 mmol1-(5-carboxy-pentyl) -2-methyl-5-sulfonato-pyridinium betaine areconverted according to the general directions given above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water.

[0069] 10% yield, MS (ESI+): 487 (M+H⁺) and 509 (M+Na⁺), UV-Vis (inwater): λ_(max) 560 nm, λ_(em) 690 nm

EXAMPLE 13

[0070]1-(5-carboxy-pentyl)-4-[(E)-2-(6-diethylamino-benzofuran-2-yl)-vinyl]-3-sulfonato-pyridiniumbetaine

[0071] 0.2 mmol 6-diethylamino-benzofuran-2-carbaldehyde and 0.2 mmol1-(5-carboxy-pentyl) -4-methyl-3-sulfonato-pyridinium betaine areconverted according to the general directions given above. Columnchromatography: SiO₂ (RP 18), eluent: methanol/water.

[0072] 15% yield, MS (ESI+): 487 (M+H⁺) and 509 (M+Na⁺), UV-Vis (inwater): λ_(max) 530 nm, λ_(em) 730 nm

EXAMPLE 14

[0073]1-(5-carboxy-pentyl)-4-[(E)-2-(6-diethylamino-benzofuran-2-yl)-vinyl]-6-sulfonato-chinoliniumbetaine

[0074] 0.2 mmol 6-diethylamino-benzofuran-2-carbaldehyde and 0.2 mmol1-(5-carboxy-pentyl) -4-methyl-6-sulfonato-chinolinium betaine areconverted according to the general directions given above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water.

[0075] 18% yield, MS (ESI+): 537 (M+H⁺) and 559 (M+Na⁺), UV-Vis (inmethanol): λ_(max) 630 nm, λ_(em) 800 nm

EXAMPLE 15

[0076]1-(5-carboxy-pentyl)-2-[(E)-2-(6-diethylamino-benzofuran-2-yl)-vinyl]-3,3-dimethyl-5-sulfonato-3H-indolium betaine

[0077] 0.2 mmol 6-diethylamino-benzofuran-2-carbaldehyde and 0.2 mmol1-(5-carboxy-pentyl)-2, 3, 3-trimethyl-5-sulfonato-3H-indolium betaineare converted according to the general directions given above. Columnchromatography: SiO₂ (RP18), eluent: methanol/water.

[0078] 12% yield, MS (ESI+): 553 (M+H⁺) and 575 (M+Na⁺), UV-Vis (inwater): λ_(max) 650 nm, λ_(em) 715 nm

[0079] General Preparation Method 2 for the Dyes of Examples 16-18:

[0080] 0.2 mmol substituted aldehyde and 0.2 mmol of the correspondingsubstituted CH-acidic compound are dissolved in 5 ml methanol and 20 μlpiperidine are added thereto. The mixture is heated for 10 hours onreflux, the solvent is removed and the residue is dried in vacuo.Purification is effected chromatographically.

EXAMPLE 16

[0081]4-{7-[(5-carboxy-pentyl)-ethyl-amino]-2-oxo-2H-chromen-3-yl}-1-(3-propylsulfonato)-pyridinium betaine

[0082] 6-[ethyl-(4-formyl-3-hydroxy-phenyl)-amino]-1-hexane acid and4-ethoxycarbonylmethyl-1-(3-propylsulfonato)-pyridinium betaine arereacted according to the general method 2.

[0083] Column chromatography: SiO₂ (RP18), eluent: methanol/water.Yield: 22 mg (21%) deep red powder

[0084] MS (ESI−): 501 [M]⁻; 1003 [2M−H]⁻; MS (ESI+): 503 [M+H]⁺; 525[M+Na]⁺; 547 [M−H+2Na]⁺, UV-Vis (in water); λ_(max) 482 nm, λ_(em) 560nm, ε=48,000 1/mol*cm

EXAMPLE 17

[0085] 1-(5-carboxy-pentyl)-4-[5, 7, 7-trimethyl-2-oxo-8-(3propylsulfonato)-7, 8-dihydro-2H-1-oxa-8-aza-anthracene-3-yl] pyridiniumbetaine

[0086] 3-(6-formyl-7-hydroxy-2, 2,4-trimethyl-2H-chinolin-1-yl)-1-propanesulfonate Na salt andN-(6-carboxyethyl-hexyl)-4-pyridinium-ethyl acetate-mesylate are reactedaccording to the general method 2.

[0087] Column chromatography: SiO₂ (RP18), eluent: methanol/water.Yield: 18 mg (15%) deep red powder

[0088] MS (ESI−): 553 [M]⁻, MS (ESI+): 577 [M+Na]⁺; 599 [M−H+2Na]⁺,UV-Vis (in water): λ_(max) 493 nm, λ_(em) 585 nm, ε=40,000 1/mol*cm

EXAMPLE 18

[0089]2-{7-[(5-carboxy-pentyl)-ethyl-amino]-2-oxo-2H-chromen-3-yl}-1-(3-propylsulfonato)-pyridiniumbetaine

[0090] 3-(6-formyl-7-hydroxy-2, 2, 4-trimethyl-2H-quinoline-1-yl)1-propanesulfonate Na salt andN-(3-propylsulfonato)-2-pyridinium-acetonitrile betaine are convertedaccording to the general method 2. The raw product is boiled 3 hoursunder reflux in a mixture of 5 ml water and 0.2 ml concentrated HCl. Themixture is neutralised by the addition of diluted NaOH, the solventremoved and the residue purified chromatographically.

[0091] Column chromatography: SiO₂ (RP18), eluent: methanol/water.Yield: 45 mg (48%) orange yellow powder

[0092] MS (ESI−): 501 [M−H]^(—); 523 [M-2H+Na]⁻, MS (ESI+): 525 [M+Na]⁺;547 [M−H+2Na]⁺, UV-Vis (in water): λ_(max) 440 nm, λ_(em) 494 nm,ε=40,000 1/mol*cm

[0093] General Preparation Method 3 for the Dyes of Examples 1 to 15, 19and 20:

[0094] 0.2 mmol of the substituted coumarin-3-carbaldehyde orbenzofurane-2-carbaldehyde and 0.2 mmol of the corresponding CH-acidiccompound or 0.2 mmol of the substituted 4-hydroxycoumarin as CH-acidiccompound and 0.2 mmol of the corresponding enolether or enamine areboiled in 5 ml acetanhydride on the reflux. The mixture is cooled after10 hours and diethylether is added. The resulting precipitation isisolated and purified by column chromatography.

EXAMPLE 19

[0095]3-(3-carboxxy-propyl)-2-[4-(7-diethylamino-4-hydroxy-2-oxo-2H-chromen-3-yl)-buta-1,3-dienyl]-3-methyl-5-sulfonato-1-(3 propylsulfonato)-3H-indolium sodiumsalt 7-diethylamino-4-hydroxy-coumarin and 3-(3-ethoxycarbonylpropyl)-3-methyl-2-(4-phenyl-amino-buta-1,3-dienyl)-5-20sulfonato-1-(3-propylsulfonato)-3H-indolium sodium salt are reactedaccording to the general method 3.

[0096] Column chromatography: SiO₂ (RP 18), eluent: methanol/water.

[0097] Yield: 23 mg (15%) deep blue powder

[0098] MS (ESI−): 350 (M²⁻/2), UV-Vis (in water): λ_(max) 606 nm, λ_(em)667 nm

EXAMPLE 20

[0099]1-(5-carboxy-pentyl)-2-[4-(7-diethylamino-4-hydroxy-2-oxo-2H-chromen-3-yl)-buta-1,3-dienyl]-3,3-dimethyl-5-sulfonato-3H-indolium betaine

[0100] 7-diethylamino-4-hydroxy-coumarin and1-(5-carboxy-pentyl)-3,3-dimethyl-2-(4-phenyl-amino-buta-1,3-dienyl)-5-sulfonato-3H-indoliumbetaine are reacted according to the general method 3.

[0101] Column chromography: SiO₂ (RP18), eluent: methanol/water. Yield20 mg (15%) deep blue powder

[0102] MS (ESI−): 621 (M⁻); 643 [M-2H+Na]⁻, MS (ESI+); 667 [M−H+2Na]⁺,UV-Vis (in water): λ_(max) 606 nm, λ_(em) 665 nm, s=180,000 1/mol*cm

1. Compound on the basis of polymethines having one of the generalformulas I-III

wherein R¹-R¹² are the same or different and represent in each casehydrogen, chlorine, bromine, alkyl, aryl, heteroaryl, cycloalkyl,heterocycloalkyl, alkyloxy, alkylmercapto, aryloxy, arylmercapto,heteroaryloxy, heteroarylmercapto or cyano groups, one or morealkyl-substituted or cyclic amino functions, each having at most 12carbon atoms, one or more hydroxy functions, R¹ and R² and/or R³ and R⁴and/or R⁷ and R⁵ and/or R⁹ and R¹⁰ can form additional aliphatic oraromatic rings, and/or two adjacent groups R⁵ and R⁶ and/or R¹¹ and R¹²together can form one or more aliphatic or aromatic rings, with theproviso that at most 2 additional rings are present, one or more of thesubstituents R¹-R¹² represent a solubilizing or ionizable or ionizedsubstituent of the group SO₃ ⁻, PO₃ ²⁻, CO₂ ⁻, O⁻, NR₃ ⁺, cyclodextrinand sugar, these substituents being unchanged or bound to the actualchromophore via an aliphatic or heteroaliphatic or cyclic spacer group,at least one of the substituents R¹-R¹² stands for a reactive group ofthe type isocyanate, isothiocyanate, hydrazine, amine, mono- anddichloro- or mono- and dibromotriazine, aziridine, sulfonyl halide, N—hydroxysuccinimide ester, imido ester, glyoxal or aldehyde or maleimideor iodoacetamide and phosphoramidite, said substituents being unchangedor bound to a basic chromophore via an aliphatic, heteroaliphatic, orcyclic spacer group, X-Y together can represent O, S, Se, Te or thestructural element (CR₂)_(n), NR or SO₂, wherein R represents equal ordifferent of the functions of R¹-R¹², and n can stand for an integerfrom 1-4, X-Y can stand for the structural elements —CR₂—O—, —O—CR₂—,—CO—O—, —O—CO—, —CO—NR— or —NR—CO—, wherein R represents equal ordifferent groups of R¹-R², Z represents the group (CR₂)_(p), wherein Rrepresents equal or different groups of R¹ to R¹², —(CH₂)_(r)—COOH or—(CH₂)_(r)—SO₃H, or their dissociable salts, p is an integer of 1 to 4and r is an integer of 1 to 7, or a combination of any of these groups,and m is zero or an integer from 1 to
 3. 2. A compound according toclaim 1 for use as a dye for the optical marking of proteins, nucleicacids, oligomers, DNA, RNA, biological cells, lipids, mono-, oligo- andpolysaccharides, ligands, receptors, polymers, pharmaceuticals orpolymer particles in immunological tests, hybridization methods, DNAsequencing, chromatographic or electrophoretic methods, inhigh-throughput screening and the analysis of receptor-ligandinteractions on a microarray.
 3. A process for the determination ofproteins, nucleic acids, oligomers, DNA, RNA, biological cells, lipids,polymers, pharmaceuticals or polymer particles, wherein the functionalgroups of the compounds according to claim 1 are coupled by covalentbonds to a HO, H₂N or HS function of the substances to be determined. 4.A process according to claim 3, wherein the coupling reaction is carriedout in an organic or aqueous medium.
 5. A process according to claim 3,wherein the compound to be covalently coupled is selected to exhibitfluorescent properties.
 6. A compound according to claim 1, wherein thealiphatic or heteroaliphatic spacer group consists of a structuralelement —[(CH₂)_(a)—W—(CH₂)_(b)]_(c)—, wherein W represents equal ordifferent of the functions CR₂—, O—, S—, SO₂—, SO₂NH—, NR—, CO₂—, orCONR—, R represents equal or different groups of R¹-R¹², and a and beach represent the same or different integers in the range from 0-18,while c represents an integer from 1 to
 18. 7. A compound according toclaim 1, wherein X-Y represents the structural element —CO—O— or —O—. 8.A compound according to claim 1, wherein R⁵ represents H, OH or alkoxyand/or R¹¹ represents carboxyalkyl.
 9. A compound according to claim 1,wherein the carboxy group in R¹¹ is in ω-position and contains at most 6carbon atoms.
 10. A compound according to claim 1, wherein m is
 1. 11. Acompound according to claim 10, wherein Z is C(CH₃)₂.
 12. A compoundaccording to claim 1, wherein R² is an amino residue, bound to thenucleus by a nitrogen atom which may be part of a ring together with R¹and/or R³.
 13. A compound according to claim 12, wherein R² contains atmost 8 carbon atoms.
 14. A compound according to claim 13, wherein R²contains a carboxyalkyl or a sulfonic acid alkyl group.
 15. A compoundaccording to claim 1, wherein up to 2 of R¹ to R¹² are or contain a SO₃⁻group.
 16. A compound according to claim 15, wherein one of R⁸ to R¹¹is or contains a SO₃ ⁻group.
 17. A compound according to claim 1,wherein a carboxyalkyl group being present as R¹¹ or contained in R² isan ω-carboxyalkyl having 6 carbon atoms.
 18. A compound according toclaim 3 for use in fluorescenceoptical measurement procedures.